CN114621936A - Protein compositions and uses thereof - Google Patents

Protein compositions and uses thereof Download PDF

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CN114621936A
CN114621936A CN202210197701.3A CN202210197701A CN114621936A CN 114621936 A CN114621936 A CN 114621936A CN 202210197701 A CN202210197701 A CN 202210197701A CN 114621936 A CN114621936 A CN 114621936A
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CN114621936B (en
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张晓洁
陈体
罗世林
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Second Xiangya Hospital of Central South University
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    • A61K38/443—Oxidoreductases (1) acting on CH-OH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
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    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
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    • C12Y102/0101—Acetaldehyde dehydrogenase (acetylating) (1.2.1.10)
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Abstract

The application discloses a protein composition and application thereof, wherein the protein composition comprises a first type of recombinant protein and a second type of recombinant protein, wherein the first type of recombinant protein is used for catalyzing ethanol to be oxidized into acetaldehyde, and the second type of recombinant protein is used for catalyzing acetaldehyde to be oxidized into acetic acid, so that the metabolism of ethanol is realized.

Description

Protein compositions and uses thereof
Technical Field
The application relates to the technical field of medicines, in particular to a protein composition and application thereof.
Background
Wine culture, as a special cultural form, holds a special position in the traditional culture of countries in the world, especially China. However, as the consumption of drinking wine increases, various negative consequences associated with drinking are also becoming more prominent as a serious public health problem. The world health organization reports that the harmful use of alcohol causes 330 million deaths each year worldwide, with approximately 5.1% of the worldwide disease and injury burden being caused by alcohol consumption. Alcohol impairs human health, such as gastrointestinal disorders, liver disorders, neurological and psychiatric disorders, cardiovascular disorders, and the like. Wherein, the damage of the alcohol to the nervous system, such as violent attack behavior, antisocial behavior, decreased accuracy of alert response speed and the like caused by cortical deshibition, impulse control obstacle, cognitive impairment and the like, brings serious threats to self health and public safety. Approximately 20-30% of alcohol abusers develop Alcoholic Liver Disease (ALD), including fatty liver, alcoholic hepatitis, cirrhosis, liver fibrosis and liver cancer. ALD is becoming one of the leading causes of morbidity and mortality worldwide. Meanwhile, studies prove that some emotional problems such as depression and alcohol use have a very complex relationship, on one hand, depressed mood is a precursor factor of harmful use of alcohol, and on the other hand, serious depression caused by long-term use of alcohol further aggravates alcohol abuse. In conclusion, alcohol use-related disorders are a problem of complex etiology and involving dysfunction of multiple organ systems.
Recent studies have found that the intestinal flora plays an important role in bidirectional regulation in alcohol use disorders, ALD and depression. On one hand, alcohol and metabolites thereof can cause intestinal tract microecological imbalance, further cause damage of intestinal mucosa barrier, induce intestinal tract permeability to increase, and cell components of intestinal microorganisms and neurotransmitters, metabolites and the like synthesized by the intestinal microorganisms can enter blood circulation, thereby affecting the health of other organs of the organism such as brain and liver. Such as endotoxin (LPS) entering the liver through portal circulation, activation of Kupffer cells in the liver in conjunction with Toll-like receptor 4 (TLR 4) is considered to be a major factor in the pathogenesis of ALD. If intestinal microecological imbalance activates immune response, immune factors enter brain through blood circulation, damage neurons, and act on hypothalamus-pituitary-adrenal Axis (HPA Axis) to cause mood disorders such as depression. On the other hand, implantation of probiotics in the intestinal tract, such as lactobacillus, bifidobacterium and Akkermansia muciniphila (a. muciniphila, AKK), significantly ameliorates the impairment of alcohol on liver function; such as lactobacillus helveticus and akkermansia muciniphila, remarkably improve depressive-like behavior. These evidences suggest that the intestinal flora plays an important role in several aspects of alcohol use-related disorders.
However, due to the complex and diverse intestinal flora, the mechanism of action in disorders associated with alcohol use is not clear, and the mechanism of the protective action of probiotics is still under investigation. Therefore, there is a need to determine the mechanism of action of probiotics on alcohol to minimize and protect against the harm of alcohol to human body.
Content of application
The present application aims to provide a protein composition and its use, which is capable of metabolizing alcohol before it enters the human body.
In order to achieve the purpose of the application, the application provides the following technical scheme:
in a first aspect, a protein composition comprising a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises a sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and has the activity of the second type of recombinant protein.
In the present application, the aforementioned SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 is derived from the intestinal mucosa bacterium Akkermansia muciniphila (a. muciniphila, AKK), is an in vitro recombinant protein, and the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 each have the function of Alcohol Dehydrogenase (ADH) for metabolizing ethanol into acetaldehyde.
Specifically, SEQ ID NO: 1 is an Amuc 0233 protein consisting of SEQ ID NO: 7, adding histidine tag tape white and thrombin cutting sites, mutating the 42 th amino acid glycine into histidine, and mutating the 338 th amino acid lysine into cysteine for modification. SEQ ID NO: 2 is Amuc-2116 consisting of SEQ ID NO: 8, by adding histidine tag tape white and thrombin cutting sites, the 42 th amino acid glycine is mutated into histidine, and the 362 th amino acid lysine is mutated into cysteine for modification. SEQ ID NO: 3 is an Amuc-1190 protein consisting of SEQ ID NO: 9, adding histidine tag tape white and thrombin cutting sites, mutating 46 th amino acid proline into histidine, and mutating 325 th amino acid lysine into cysteine for modification.
Wherein, SEQ ID NO: 7. SEQ ID NO: 8 and SEQ ID NO: and 9, the three proteins are all proteins included in AKK bacteria.
The alcohol dehydrogenase ADH is abundantly present in human and animal livers, plants and microbial cells and can metabolize alcohol into acetaldehyde as a key enzyme for the main short-chain alcohol metabolism in organisms. Likewise, SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 by replacing, deleting or adding one or more amino acids respectively, and the derivative protein with the activity of the first type of recombinant protein also has the function of alcohol dehydrogenase.
And, the aforementioned SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 6 is also from the intestinal mucosa bacterium Akkermansia muciniphila (a. muciniphila, AKK), is an in vitro recombinant protein, and the amino acid sequence of SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6 all have the function of acetaldehyde dehydrogenase for metabolizing acetaldehyde to acetic acid.
Specifically, SEQ ID NO: 4 is an Amuc-2066 protein consisting of SEQ ID NO: 10 is obtained by adding histidine tag tape white and thrombin cutting site, and mutating the amino acid glutamic acid at the 1142 position into lysine modification. SEQ ID NO: 5 is an Amuc-0528 protein encoded by SEQ ID NO: 11, by adding histidine tag tape white and thrombin cutting sites, the 390 th amino acid glutamic acid is mutated into lysine modification. SEQ ID NO: 6 is an Amuc-1265 protein consisting of the amino acid sequence of SEQ ID NO: 12, adding histidine tag tape white and thrombin cutting sites, and mutating the 332 th amino acid threonine into lysine modification.
Wherein, SEQ ID NO: 10. SEQ ID NO: 11 and SEQ ID NO: 12 three proteins are all proteins included in AKK bacteria.
Acetaldehyde dehydrogenase (ALDH) exists in the liver of human body and animals, and is responsible for catalyzing the reaction of oxidizing acetaldehyde into acetic acid after alcohol enters the human body (acetaldehyde is obtained by catalyzing the ethanol under the action of the alcohol dehydrogenase), acetaldehyde generated under the action of the alcohol dehydrogenase is further converted into harmless acetic acid under the catalysis of the aldehyde dehydrogenase when alcohol is metabolized in the liver of the human body, and the acetic acid is finally decomposed into carbon dioxide and water to complete alcohol metabolism. Likewise, SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by replacing, deleting or adding one or more amino acids respectively, and the derivative proteins with the activity of the second type of recombinant protein have the function of acetaldehyde dehydrogenase.
In a second aspect, there is provided the use of a protein composition in the manufacture of a product for metabolizing ethanol;
the protein composition comprises a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and having the activity of said second type of recombinant protein.
Further, the first type of recombinant protein is used for metabolizing ethanol into acetaldehyde;
the second type of recombinant protein is used to metabolize the acetaldehyde obtained into acetic acid.
In a third aspect, there is provided the use of a host expressing a protein composition in the manufacture of a product for metabolizing ethanol.
Wherein the protein composition comprises a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NOs: 1. SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and having the activity of said second type of recombinant protein.
Wherein, the host is a natural host or a recombinant host prepared by genetic engineering means.
In some embodiments, the native host is a microorganism that is naturally occurring and capable of expressing both the first type of recombinant protein and the second type of recombinant protein. For example, the host expressing the first and second recombinant proteins is a microorganism of Verrucomicrobia family. In some embodiments, the microorganism of the Verrucomicrobia family is AKK bacteria. It is understood that the Akkermansia muciniphila in the present application includes not only a. muciniphila but also microorganisms having more than 90% similarity in nucleic acid sequence, particularly greater than 95% similarity to a. muciniphila, which is called Akkermansia-like.
In other embodiments, the host is a recombinant host expressing a first type of recombinant protein and a second type of recombinant protein. The recombinant host is a microorganism or a cell strain; the recombinant host expresses a protein composition which comprises a first type of recombinant protein and a second type of recombinant protein. For example, by genetic engineering means, a host that does not express the first type of recombinant protein or the second type of recombinant protein produces a large amount of the first type of recombinant protein or the second type of recombinant protein; or the gene engineering means is used for carrying out substitution, addition and deletion of single amino acid or a plurality of amino acid sites, so that the functions of the first type of recombinant protein and the second type of recombinant protein are more obvious or inactivated. Such genetic engineering means include, but are not limited to, ZFNs, TALENs, or CRISPR/Cas 9. Hosts include, but are not limited to, bacteria, fungi, plant cells, or animal cells. Wherein the bacteria are selected from Escherichia coli, such as Escherichia coli BL21(DE3), and the mammalian cells are selected from HEK293 cell line.
In a fourth aspect, a nucleic acid vector is provided that includes a backbone vector and a polynucleotide encoding the protein composition.
In a preferred embodiment, the backbone vector is Pet-28 or pcDNA4a (+).
In a fifth aspect, there is provided a product comprising at least one of the following components:
a. a protein composition comprising a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. the amino acid sequence of SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and has the activity of the second type of recombinant protein.
b. A host expressing the protein composition of a;
c. b a fermentation product of said host;
d. b an extract of said host and/or c said fermentation product;
e. other substances having biological activity of metabolizing alcohol.
Further provides an application of the product in preparing anti-alcohol drugs/health care products.
In a sixth aspect, there is provided a pharmaceutical composition comprising a protein composition as an active ingredient, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant and/or diluent;
the protein composition comprises a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and has the activity of the second type of recombinant protein.
Compared with the prior art, the method has the following beneficial effects:
the application provides a protein composition and application thereof, wherein the protein composition comprises a first type of recombinant protein and a second type of recombinant protein, wherein the first type of recombinant protein is used for catalyzing ethanol oxidation to acetaldehyde, and the second type of recombinant protein is used for catalyzing acetaldehyde oxidation to acetic acid, so that the metabolism of ethanol is realized, and ethanol dehydrogenase and acetaldehyde dehydrogenase in a human body are replaced.
Drawings
FIG. 1 is an analysis of the effect of AKK bacteria on alcohol consumption;
FIGS. 2A and 2B are graphs showing the analysis of AKK bacteria for improving anxiety in alcohol model mice;
FIGS. 3A-3D are graphs of the analysis of the effect of AKK bacterial intervention on mouse serum liver function indices in a mouse chronic alcohol gavage model;
FIGS. 4A and 4B are graphs of the effect of AKK bacteria on serum alcohol concentration versus intestinal permeability change;
FIGS. 5A-5G are graphs of intestinal permeability assays of mice in the AKK bacteria intervention group;
FIGS. 6A-6C are graphs showing the analysis of the time spent in vivo quantifying the concentration of alcohol in serum by AKK bacteria;
FIG. 7 is a graph showing the change in serum alcohol concentration before and after the gavage treatment with AKK bacteria.
Detailed Description
In order to make the purpose, technical solutions and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In the description of the present application, it is to be understood that the terms "first", "second" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying any number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present application, "a plurality" means two or more unless otherwise specified.
Examples
The test materials used in this embodiment are all commercially available products, and are all commercially available.
The various bacteria information used in this example is as follows:
sources of akkermansia muciniphila: zhengzhou Jingsi waffle, Inc., originally sourced from (ATCC, BAA-835), with 5 generations of working seed lots.
The present example provides a protein composition comprising a first type of recombinant protein and a second type of recombinant protein. Wherein the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein. The second type of recombinant protein comprises the amino acid sequence as shown in SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and has the activity of the second type of recombinant protein. Wherein, SEQ ID NO: 1 to SEQ ID NO: 6 are represented by SEQ ID NO: 7 to SEQ ID NO: 12 by corresponding site modification, and SEQ ID NO: 7 to SEQ ID NO: 12 is a protein included in AKK bacteria.
The protein composition includes, but is not limited to, a culture solution of akkermansia muciniphila, an extract solution of akkermansia muciniphila, a purified solution of recombinant protein, and the like. The recombinant protein can also be prepared by introducing the gene sequence into engineering bacteria by conventional technical means, fermenting, separating and purifying. A specific method for preparing recombinant protein comprises the following steps:
amplifying a gene sequence for coding recombinant protein by PCR, cloning the sequence to a pET-28a vector by a method of enzyme digestion and enzyme ligation, constructing an expression plasmid of the recombinant protein with an N-end fused with a 6 XHis tag, performing heterologous expression by using an engineering bacterium of escherichia coli BL21(DE3), culturing the strain on an LB + KanR resistant plate overnight, selecting a monoclonal antibody, transferring the monoclonal antibody into 5ml of LB culture solution containing 100 mu g/ml KanR, culturing at 37 ℃ and 180rpm for 4 hours, transferring 1:100 into a new culture medium at a middle index (0D600 ═ 0.6), continuously culturing at 180rpm for 4 hours, adding IPTG to a final concentration of 0.5-2 mM, and inducing at 28 ℃ and 180rpm overnight. The bacterial solution was centrifuged at 8,000rpm for 5 minutes to precipitate cells, and the cells were stored at-20 ℃.
The thalli is evenly mixed in 10 times volume of ultrasonic lysate, 4ml of PMSF containing lysozyme, DNase/RNase and 1mM of final concentration is added, the mixture is evenly mixed, the mixture is placed on ice for ultrasonic 90min to be cracked and broken until the solution is completely clear, the cell lysate is centrifuged at 12,000rpm, 30min and 4 ℃, supernatant is collected and placed on ice to obtain recombinant protein, the recombinant protein is obtained through Ni-16NTA His-Bind resin affinity chromatography purification, the split charging is carried out, and the recombinant protein is prepared into proper concentration when being stored at minus 80 ℃ for standby.
The alcohol metabolism ability of AKK bacteria expressing the protein composition is examined below.
As shown in figure 1, a mouse alcohol diet feeding model is established, and AKK bacteria are found to influence the daily alcohol intake of mice.
Specifically, experiments show that the AKK bacteria or a solvent (glycerol saline) contrast gastric perfusion intervention is started on the 14 th day of the model building of the mice fed with the alcohol diet, the intake of the mice in the AKK bacteria intervention group to alcohol per day is obviously reduced from the fourth day after the gastric perfusion, and then the autonomous alcohol consumption of the mice is obviously reduced compared with that of a contrast group in the whole alcohol diet model building process, so that the AKK bacteria can reduce the damage of the alcohol to the organism by reducing the autonomous alcohol consumption of the mice, and further reduce the damage of the alcohol to the liver tissues of the mice. Therefore, it is thought that AKK bacteria can affect the daily alcohol intake in mice.
As shown in FIGS. 2A and 2B, AKK bacteria are found to improve anxiety and depression behaviors of the alcohol model mice through experiments.
Specifically, the ALD mouse is subjected to AKK bacteria intragastric administration intervention, Forced Swimming (FST) and open field experiments (OFT) find that the AKK bacteria remarkably relieves depression-like behavior of the alcohol model mouse and improves anxiety-like behavior of the alcohol model mouse. This suggests that AKK bacteria may be a therapeutic target for improving anxiety depression or anxiety depression disorder caused by alcohol.
As shown in FIGS. 3A-3B, a chronic alcohol gavage model was established, and experiments found that AKK bacteria had a protective effect on liver injury caused by alcohol.
After the AKK bacteria is subjected to intragastric administration intervention for 4 weeks, alcohol chronic intragastric administration is performed, the influence of the AKK bacteria intervention on serum liver function indexes of the mice is analyzed, the liver injury caused by the AKK bacteria intervention group is found to be lighter than that of a simple alcoholic liver disease group, and the elevation degree of ALT and AST is also reduced. This indicates that AKK bacteria has a protective effect on liver damage caused by alcohol.
As shown in FIGS. 4A-4B, it was found that the effect of AKK bacteria on serum alcohol concentration was independent of intestinal permeability changes.
Specifically, when the mice are fed with the alcohol diet, the intestinal permeability of the mice in an AKK bacteria intervention group is not obviously changed, but the alcohol concentration in serum is obviously reduced by adopting the contrast gavage intervention of AKK bacteria or a solvent (glycerol saline) for the mice, which shows that the influence of the AKK bacteria on the alcohol concentration in the serum is unrelated to the change of the intestinal permeability, and the AKK bacteria can play an important role in the alcohol metabolism process.
As shown in FIGS. 5A-5G, it was experimentally found that the effect of AKK bacteria on serum alcohol concentration was prior to the entry of alcohol into the intestinal wall of the host and was not associated with host cell ADH and ALDH activity.
Performing alcohol chronic gavage for 8 weeks after performing control gavage pretreatment on AKK bacteria or a solvent (glycerol saline) for 4 weeks, performing alcohol gavage for 30 minutes in the last day, and detecting alcohol concentration, ADH activity and ALDH activity in blood, intestinal tract tissue and liver respectively. The results suggest that the alcohol concentration in blood, intestinal tract tissue and liver of the mice pretreated by AKK bacteria is reduced remarkably, while the ADH activity and ALDH activity in the intestinal tract tissue and the liver tissue are not different remarkably, which indicates that the influence of the AKK bacteria on the alcohol concentration in serum is probably before the alcohol enters the intestinal wall of the host and is not related to the ADH and ALDH activity of the host cells.
As shown in FIGS. 6A-6C, it was found that AKK has been colonized in vivo for a sufficiently long time to have an effect on the concentration of alcohol in serum.
Further research shows that after long-enough permanent planting, the AKK bacteria can obviously reduce the alcohol concentration in blood of a mouse after single alcohol gavage, and the akkermansia muciniphila has no obvious influence on the alcohol concentration in serum after two times of gavage or even after 15 times of gavage. It can be seen that AKK needs to be colonized in vivo for a long enough time to affect the effect of alcohol concentration in serum.
As shown in FIG. 7, the experiment shows that the AKK bacteria has an influence on the concentration of alcohol in the serum of the rat after a single alcohol gavage 21 days.
Specifically, the rats are subjected to 21-day AKK bacteria intragastric administration, tail vein blood sampling is respectively carried out before single-time alcohol intragastric administration and after 5 minutes, 10 minutes, 30 minutes, 60 minutes and 120 minutes of intragastric administration, and alcohol concentration change is measured, so that the result indicates that the alcohol concentration in serum of the rats subjected to AKK bacteria pretreatment is maintained at a remarkably reduced level after single-time alcohol intragastric administration, and the effect of remarkably reducing the alcohol concentration in the serum by using the AKK bacteria fixed planting is verified again.
Therefore, the AKK bacteria expressing the protein composition provided by the embodiment can complete alcohol metabolism before alcohol enters a human body, wherein the first type of recombinant protein is used for catalyzing ethanol oxidation to acetaldehyde and has the function of ethanol dehydrogenase, and the second type of recombinant protein is used for catalyzing acetaldehyde oxidation to acetic acid and has the function of acetaldehyde dehydrogenase, so that alcohol in a digestive tract before entering the human body is effectively metabolized, and human damage, emotional problems and alcohol use related obstacles caused by alcohol are reduced.
Further, the present embodiment also provides a product, which includes at least one of the following components:
a. a protein composition comprising a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences set forth in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and has the activity of the second type of recombinant protein.
b. A host expressing the protein composition of a;
c. b a fermentation product of said host;
d. b an extract of said host and/or c said fermentation product;
e. other substances having biological activity of metabolizing alcohol.
8. The use of the product of claim 7 in the preparation of anti-hangover drugs/health products.
And, further provides a pharmaceutical composition comprising the protein composition as an active ingredient, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant and/or diluent.
Correspondingly, the pharmaceutical composition also has the alcohol metabolism function.
Thus, the user takes the pharmaceutical composition before drinking to reserve a certain amount of the pharmaceutical composition in the digestive tract and form a protective barrier. When a user drinks and the alcohol reaches the digestive tract, the pharmaceutical composition can effectively metabolize the alcohol so as to reduce the absorption of the human body to the alcohol, thereby avoiding the physical and psychological trauma of the alcohol to the user.
All the above optional technical solutions can adopt any combination to form the optional embodiments of the present application, that is, any multiple embodiments can be combined, so as to obtain the requirements for coping with different application scenarios, which are within the protection scope of the present application and are not described herein any more.
It should be understood that the above-mentioned embodiments are merely preferred embodiments of the present application and are not intended to limit the present application, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.
Sequence listing
<110> Xiangya II Hospital of Zhongnan university
<120> protein compositions and uses thereof
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Gln Ala Gly Pro Gly Leu Glu Leu Met Asp Val Pro Met Pro Glu Val
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Gly Pro Asn Asp Val Leu Ile Lys Ile His Lys Thr Ala Ile Cys His
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Thr Asp Leu His Ile Trp Asn Trp Asp Lys Trp Ala Gln Gln Thr Ile
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Pro Val Gly Met His Val Gly His Glu Phe Cys Gly Val Ile Glu Ser
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Val Gly Ser Ser Val Thr Glu Tyr Lys Pro Gly Glu Ile Val Ser Gly
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Glu Gly His Ile Val Cys Gly His Cys Arg Ser Cys Arg Ser Gly Gln
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Lys His Leu Cys Pro Asn Thr Lys Gly Val Gly Val Asn Arg Pro Gly
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Cys Phe Ala Glu Tyr Leu Ser Ile Pro Gln Asp Asn Val Val Arg Ile
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His Lys Ser Ile Pro Met Glu Ile Ala Ser Ile Phe Asp Pro Leu Gly
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Asn Ala Val His Thr Ala Leu Ser Trp Asp Leu Val Gly Glu Asp Val
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Leu Ile Thr Gly Ala Gly Val Ile Gly Cys Met Ala Ala Ala Val Cys
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Lys Lys Ala Gly Ala Lys Thr Val Val Ile Thr Asp Ile Asn Asp Phe
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Arg Leu Gly Leu Ala Lys Thr Leu Gly Ala Asp Arg Thr Val Asn Val
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Thr Arg Glu Lys Leu Glu Asp Val Met Lys Glu Leu Glu Met Thr Glu
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Gly Phe Asp Val Cys Leu Glu Met Ser Gly Ala Pro Ser Cys Leu Lys
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Asp Ile Ile Asp Asn Ser Arg Asn Gly Ala Asn Ile Ser Leu Leu Gly
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Ile Gln Pro Asp Gly Ser Ser Ile Glu Trp Asn Lys Phe Ile Trp Lys
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Gly Leu Lys Met Lys Gly Ile Tyr Gly Arg Glu Ile Phe Glu Thr Trp
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His Lys Met Asp Ser Met Ile Arg Ser Gly Leu Asn Val Ala Pro Ile
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Thr Ala Pro Lys Thr Phe Glu Ile Arg Glu Tyr Pro Ile Pro Ala Ile
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Gly Asp Asp Glu Met Leu Ile Lys Val Glu Ala Cys Gly Val Cys His
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Thr Asp Gly His Glu Tyr Asn Arg Asp Pro Phe Gly Leu Cys Pro Val
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Val Leu Gly His Glu Gly Thr Gly Glu Ile Val Ala Met Gly Arg Asn
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Ile Thr Lys Asp Thr Ala Gly Asn Pro Val Ala Leu Gly Asp Lys Ile
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Val Thr Cys Ile Ile Pro Cys Gly Thr Cys Asp Ala Cys Leu Asn Thr
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Pro Ala Arg Thr Asn Leu Cys Glu Asn Val Gly Val Tyr Gly Leu Met
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Pro Asp Asp Asp Val His Leu Asn Gly Tyr Phe Gly Glu Tyr Leu Val
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Ile Arg Lys Gly Ser Thr Phe Phe Asn Val Ser Gly Met Thr Leu Asp
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Gln Arg Ile Leu Val Glu Pro Ala Ala Val Val Val His Ser Leu Glu
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Arg Ala Lys Ser Thr Gly Leu Leu Lys Phe Asn Ser Val Val Leu Val
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Gln Gly Cys Gly Pro Ile Gly Leu Leu Gln Ile Ala Thr Leu Arg Thr
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Leu Gly Ile Glu Thr Ile Ile Ala Val Asp Gly Asn Asp Ser Arg Leu
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Glu Leu Ala Arg Glu Met Gly Ala Ser Arg Thr Tyr Asn Phe Thr Arg
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Tyr Ala Asp Leu Asn Glu Leu Leu Asp Ala Val Lys Lys Asp Asn Gly
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Gly Arg Leu Ala Asp Phe Val Phe Gln Cys Thr Gly Val Gly Lys Ala
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Gly Ala Asn Ala Trp Lys Phe Val Lys Arg Gly Gly Gly Leu Cys Glu
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Val Gly Phe Phe Met Asp Gly Gly Glu Ser Val Ile Asn His His Tyr
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Asp Leu Cys Asn Lys Glu Val Thr Ala Val Gly Ser Trp Val Tyr Ser
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Pro Gln Asp Tyr Pro Thr Thr Phe Asp Phe Leu Lys Arg Ala Tyr Gly
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Ile Gly Leu Pro Leu Thr Lys Leu Ile Ser His Arg Phe Lys Leu Asp
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Ile Ala Val Ile Cys Asn Cys Ser Lys Asn Ile
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Glu Cys Ser Met Lys Pro Gln Glu Val Leu Glu Tyr Val Ser Gly Pro
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Ile Pro Val Pro Glu Glu Gly Glu Val Leu Val Arg Met Lys Ala Ala
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Pro Ile Asn His Ala Asp Ile Asn Phe Val Gln Gly Val Tyr Gly Leu
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Lys Pro Val Leu Pro His Ser Arg Ala Gly Leu Glu Gly Cys Gly Val
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Val Gln Glu Ser Arg Ala Ala Gly Phe Arg Glu Gly Asp Glu Val Ile
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Leu Leu Arg Gly Val Gly Ser Trp Ser Glu Tyr Val Ala Val Pro Ser
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Val Asn Val Met Lys Leu Pro Val Lys Val Asp Pro Val Gln Ala Ala
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Met Leu Lys Val Asn Pro Leu Thr Ala Leu Arg Met Leu Glu Gly Phe
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Val Ser Leu Glu Pro Gly Asp Trp Leu Val Gln Asn Ala Ala Asn Ser
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Gly Val Gly Arg Cys Ile Ile Gln Leu Ala Arg Glu Met Gly Val Lys
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Thr Val Asn Phe Val Arg Arg Pro Asp Glu Leu Arg Asp Glu Leu Thr
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Ala Leu Gly Ala Asp Leu Val Val Gly Glu Asp Asp Gly Asp Val Val
195 200 205
Lys Asn Thr Leu Ala Arg Leu Asp Gly Lys Arg Pro Val Leu Ala Ser
210 215 220
Asn Ala Val Gly Gly Glu Ser Ala Leu Arg Leu Met Asp Met Leu Ala
225 230 235 240
Pro Gly Gly Ser Met Val Thr Tyr Gly Ala Met Ser Arg Lys Ser Ile
245 250 255
Lys Val Pro Asn Gly Phe Leu Ile Phe Lys Gly Ile Lys Leu Glu Gly
260 265 270
Leu Trp Val Thr Gln Trp Leu Lys Asn Ala Pro Val Ser Glu Ile Glu
275 280 285
Ala Ala Tyr Glu Lys Leu Ala Arg Leu Met Ala Asp Gly Arg Leu Lys
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Gln Ala Val Asp Thr Val Tyr Pro Leu Ser Asp Val Arg Lys Ala Val
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Glu Lys Ala Gln Glu Glu Phe Arg Ser Gly Lys Cys Val Leu Ser Met
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Asp Cys Ala
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Leu Val Pro Arg Gly Ser Met Thr Asp Ser Ser Ile Pro Asp Met Met
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Ala Glu Ala Arg Arg Gly Gln Trp Thr Asp Gln Gln Leu Ala Ala Lys
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Ala Val Glu Leu Ala Glu Ser Ile Leu Lys Gln Ser Asn Ala Gly Met
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Arg Gly Lys Glu Lys Arg Gln Ala Gln Gln Met Glu Arg Met Met Asn
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Asp Pro Ala Gly Lys Ala Phe Thr Leu Ala Leu Ala Asp Arg Val Phe
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Arg Pro Ser Ser Pro Val Arg Gly Ala Glu Leu Phe Arg Tyr Leu Leu
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Asp Gly Tyr Gly Val Pro Arg Tyr Leu Ser Ala Ala Asp Arg Phe Ala
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Ile Lys Met Gly Gly Arg Phe Ser Ala Gln Phe Pro Gly Val Val Met
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Pro Val Ile Thr Ser Gln Leu Arg Lys Glu Ser Ser Asn Val Ile Leu
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Pro Ala Glu Asp Gly Lys Leu Arg Pro His Leu Arg Arg Arg Arg Lys
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Gly Gly Ile Arg Met Asn Ile Asn Gln Leu Gly Glu Ala Ile Leu Gly
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Glu Ser Glu Ala His His Arg Leu Gln Gln Val Val Asp Arg Leu Thr
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Asp Lys Asp Cys Asp Tyr Ile Ser Val Lys Ile Ser Ala Ile Phe Ser
195 200 205
Gln Ile His Leu Val Ala Phe Glu Glu Thr Val Lys Leu Ile Gln Glu
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Arg Leu Arg Ile Leu Tyr Arg Ala Ala Ile Thr Asn Ala Val Thr Leu
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Pro Asp Gly Ser Arg Lys Pro Lys Phe Val Asn Leu Asp Met Glu Glu
245 250 255
Tyr Arg Asp Leu His Leu Thr Ala Glu Ala Phe Lys Arg Thr Leu Met
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Glu Asp Glu Phe Met Gln Leu Glu Ala Gly Ile Val Leu Gln Ala Tyr
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Leu Pro Asp Ser Trp Glu Glu Gln Met Lys Leu Cys Ala Trp Ala Lys
290 295 300
Glu Arg Val Glu Gln Gly Gly Ala Arg Ile Lys Ile Arg Leu Val Lys
305 310 315 320
Gly Ala Asn Leu Ala Met Glu Lys Val Glu Ala Ser Met His Gly Trp
325 330 335
Ala Gln Ala Pro Tyr Gly Thr Lys Ala Gln Val Asp Ala Asn Tyr Lys
340 345 350
Arg Met Leu His Tyr Gly Cys Met Pro Asp Asn Ala Arg Tyr Val Gln
355 360 365
Phe Gly Val Ala Ser His Asn Leu Phe Asp Leu Cys Tyr Ala Met Leu
370 375 380
Leu Arg Glu Arg Glu Gly Val Arg Asp Gln Val Glu Phe Glu Met Leu
385 390 395 400
Glu Gly Met Ala Asn His Gln Ala Arg Val Ile Arg Gln Ala Ala Asp
405 410 415
Gly Leu Leu Leu Tyr Ala Pro Val Val Leu Lys Glu Asp Phe His Ser
420 425 430
Ala Ile Ala Tyr Leu Val Arg Arg Leu Asp Glu Asn Thr Ser Glu Glu
435 440 445
Asn Phe Leu His Asp Leu Phe Gly Met Thr Pro Gly Ser Arg Ser Trp
450 455 460
Glu Val Gln Lys Lys Arg Phe Leu Lys Ala Cys Gln Glu Lys Asp Glu
465 470 475 480
Val Lys Tyr Gly Pro Asn Arg Thr Gln Asn Arg Ala Ala Asp Pro Ile
485 490 495
Gln Pro Ser His Tyr Arg Asp Ala Phe Ala Asn Glu Arg Asp Thr Asp
500 505 510
Trp Ser Leu Arg Gln Asn Ala Glu Trp Ile Asn Gly Leu Ile Ala Ala
515 520 525
Glu Lys Glu Lys Ser Gly Glu Glu Ile Pro Leu Val Ile Asp Gly Glu
530 535 540
Glu Ile Thr Thr Asn Leu Trp Gly Val Gly Arg Asp Pro Ser Arg His
545 550 555 560
Asn Glu Val Ser Tyr Lys Phe Ala Tyr Ala Asp Phe Asp Gln Val Glu
565 570 575
His Ala Leu Val Thr Ala Asp Arg Val Arg Ser Ser Trp Ala Ser Lys
580 585 590
Ser Ile Gly Glu Arg Ala Glu Ile Leu His Arg Ala Ala Gln Glu Leu
595 600 605
Ser Arg Ile Arg Gly Glu Ala Ile Ala Ala Met Val Arg Asp Ala Gly
610 615 620
Lys Ala Pro Thr Glu Ala Asp Val Glu Val Ser Glu Ala Ile Asp Phe
625 630 635 640
Cys Arg Tyr Tyr Ala Glu Gly Leu Asp Arg Asp Gly Met Asn Asp Gly
645 650 655
Val Glu Met Ser Pro Leu Gly Ile Ile Cys Val Met Ser Pro Trp Asn
660 665 670
Phe Pro Phe Ala Ile Pro Thr Gly Gly Val Ala Ala Ala Leu Met Ala
675 680 685
Gly Asn Ala Val Val Phe Lys Pro Ser Glu Leu Ala Val Tyr Thr Ala
690 695 700
Trp Gln Ile Val Gln Ala Phe Trp Arg Ala Gly Val Pro Lys Asn Val
705 710 715 720
Leu Gln Phe Val Pro Met Pro Arg Asn Glu Ile Ser Cys Lys Phe Leu
725 730 735
Met Asp Pro Arg Leu Asn Gly Val Ile Met Thr Gly Ser Tyr Arg Thr
740 745 750
Gly Lys Met Leu Arg Glu Leu Arg Pro Asp Leu His Val Leu Ala Glu
755 760 765
Thr Ser Gly Lys Asp Ala Met Ile Ile Thr Ala Thr Ala Asp Pro Asp
770 775 780
Gln Ala Val Lys Asp Leu Val Lys Ser Ala Phe Gly His Ser Gly Gln
785 790 795 800
Lys Cys Ser Ala Ala Ser Val Ala Ile Val Glu Ala Ser Val Tyr Asp
805 810 815
Asn Pro Ala Phe Leu Arg Gln Leu Lys Asp Ala Ala Ala Ser Leu Lys
820 825 830
Val Gly Gly Ser Trp Glu Val Asn Ser Val Val Thr Pro Leu Ile Arg
835 840 845
Glu Pro Glu Gly Asn Leu Leu Arg Ala Leu Thr Gln Leu Glu Pro Gly
850 855 860
Glu Glu Trp Leu Leu Lys Pro Glu Pro Ser Glu Asp Asn Pro Cys Leu
865 870 875 880
Trp Ser Pro Gly Ile Arg Leu Gly Val Lys Pro Gly Ser Trp Phe His
885 890 895
Gln Thr Glu Cys Phe Gly Pro Val Leu Gly Ile Ile Arg Ala Glu Asn
900 905 910
Leu Glu Glu Ala Ile Asp Ile Gln Asn Asp Ser Glu Phe Gly Leu Thr
915 920 925
Gly Gly Leu Gln Ser Leu Asp Glu Arg Glu Ile Ala Leu Trp Lys Thr
930 935 940
Lys Val Gln Val Gly Asn Ala Tyr Ile Asn Arg Val Ile Thr Gly Ala
945 950 955 960
Ile Val Arg Arg Gln Pro Phe Gly Gly Trp Asn His Ser Ser Met Gly
965 970 975
Pro Gly Ala Lys Ala Gly Gly Lys Asn Tyr Leu Thr Met Leu Gly Ser
980 985 990
Trp Glu Glu Lys Ala Leu Pro Gln Lys Leu Arg Thr Pro Gly Glu Arg
995 1000 1005
Ile Ser Gly Leu Val Glu Lys Leu Cys Ser Glu Leu Pro Asp Cys Ala
1010 1015 1020
Lys Arg Ile Arg Ser Ala Ala Gly Ser Gln Ala Lys Trp Trp Met Glu
1025 1030 1035 1040
Glu Phe Gly Val Asp His Asp Pro Ser Arg Val Tyr Gly Glu Asn Asn
1045 1050 1055
Thr Phe Arg Tyr Ile Pro Val Lys Gly Ile Leu Ala Arg Val Glu Asn
1060 1065 1070
Met Ser Asp Asp Asp Val Ala Ile Leu Leu Leu Gly Ala Lys Leu Cys
1075 1080 1085
Gly Val Leu Leu His Leu Ser Ile Gly Thr Ser Arg Pro Trp Ile Gln
1090 1095 1100
Lys Met His Gly Tyr Tyr Ala Ser Leu Thr Val Glu Thr Glu Ala Glu
1105 1110 1115 1120
Leu Ile Gly Arg Met Pro Glu Ala Leu Pro Gly Ile Arg Phe Leu Arg
1125 1130 1135
Gly Thr Asp Ile Ser Glu Thr Leu Ala Asn Ala Ala Arg Ala Arg Asp
1140 1145 1150
Val Glu Val Leu Asp Arg Pro Val Leu Ala Asn Gly Arg Leu Glu Leu
1155 1160 1165
Leu Gly Tyr Phe Arg Glu Gln Ser Val Ser Glu Thr Val His Arg Tyr
1170 1175 1180
Gly Asn Leu Ile Pro Pro Pro Gly Ser Phe Lys Thr Asp Ser Val
1185 1190 1195
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Leu Val Pro Arg Gly Ser Met Lys Met Glu Asn Pro Leu Glu Pro Ala
1 5 10 15
Gly Arg Ala Ala Leu Ala Ala Ser Arg Glu Met Leu Ala Leu Glu Pro
20 25 30
Glu Glu Lys Ala Gln Ala Leu Arg Leu Met Ala Ala Gly Val Arg Asn
35 40 45
Ala Ala Glu Lys Ile Ile Arg Glu Asn Val Leu Asp Met Glu Glu Ala
50 55 60
Arg Lys Ser Gly Arg Asn Ser Ala Phe Leu Asp Arg Leu Leu Leu Thr
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Pro Asp Arg Val Glu His Met Ala Cys Gly Ile Glu Gln Val Ala Ala
85 90 95
Leu Pro Asp Pro Val Gly Glu Cys Ile Arg Glu Trp Thr Arg Pro Asn
100 105 110
Gly Leu Arg Ile Arg Gln Val Arg Val Pro Leu Gly Val Ile Gly Phe
115 120 125
Ile Tyr Glu Ser Arg Gly Thr Val Thr Cys Asp Ala Ala Ala Leu Cys
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Leu Lys Ser Gly Asn Ala Val Ile Leu Arg Gly Gly Ser Glu Ser Leu
145 150 155 160
Arg Thr Asn Arg Val Leu Ala Glu Thr Leu Arg Ser Ala Leu Glu Lys
165 170 175
Ser Ala Val Pro Ala Asp Ala Val Arg Leu Leu Asp Ser Gly Ser Arg
180 185 190
Asp Glu Val Arg Gln Leu Cys Glu Leu Asp Ser Cys Leu Asn Val Ile
195 200 205
Ile Pro Arg Gly Gly Lys Gly Leu Ile Arg Ala Val Thr Glu Phe Ser
210 215 220
Lys Val Pro Val Leu Lys His Leu Asp Gly Ile Cys His Val Tyr Val
225 230 235 240
Asp Ala Ala Ala Asp Leu Glu Met Ala Val Asn Val Leu Asp Asp Ala
245 250 255
Lys Thr Gln Arg Pro Gly Val Cys Asn Ala Ala Glu Thr Leu Leu Val
260 265 270
Asp Ala Ala Ala Ala Glu Arg Phe Leu Pro Met Ala Ala Glu Arg Met
275 280 285
Arg Met Arg Gly Val Glu Cys Arg Val Cys Glu Arg Ser Arg Pro Phe
290 295 300
Phe Gly Gln Glu Ala Val Pro Ala Glu Glu Arg Asp Trp Ser Thr Glu
305 310 315 320
Tyr Glu Asp Leu Ile Leu Ser Val Lys Val Val Asp Gly Val Arg Asp
325 330 335
Ala Val Glu His Ile Asn Arg Tyr Gly Ser His His Ser Asp Ser Ile
340 345 350
Ile Thr Glu Asp Lys Arg Ala Arg Asp Tyr Phe Met Asn Arg Val Asp
355 360 365
Ser Ala Cys Val Tyr His Asn Cys Ser Thr Arg Phe Ser Asp Gly Glu
370 375 380
Glu Phe Gly Phe Gly Ala Glu Ile Gly Ile Gly Thr Asp Lys Phe His
385 390 395 400
Ala Arg Gly Lys Met Ala Leu Arg Glu Leu Thr Ser Tyr Lys Tyr Val
405 410 415
Ile Glu Gly Asn Gly Gln Leu Lys Glu Pro Ser Arg Ile Pro Gly Glu
420 425 430
Ser Arg Ala
435
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Leu Val Pro Arg Gly Ser Met Asn Gln Ala Pro His Val Ala Ile Val
1 5 10 15
Gly Ala Thr Gly Ala Val Gly Val Glu Ile Leu Ser Cys Leu Glu Thr
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Arg Asn Phe Pro Val Gly Ser Leu Lys Leu Leu Ala Ser Ala Arg Ser
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Ala Gly Lys Gln Val Ala Phe Arg Gly Lys Met Leu Thr Val Glu Glu
50 55 60
Leu Thr Glu Lys Ser Phe Asp Gly Val Asp Ile Ala Leu Phe Ser Ala
65 70 75 80
Gly Gly Gly Ile Ser Leu Lys Phe Ala Pro Ile Ala Ala Ala Ala Gly
85 90 95
Cys Val Val Ile Asp Asn Ser Ser Ala Phe Arg Gln Glu Pro Asp Val
100 105 110
Pro Leu Val Val Pro Glu Ile Asn Pro Glu Ala Ala Phe Asn His Pro
115 120 125
Arg Asn Ile Ile Ala Asn Pro Asn Cys Thr Thr Ile Ile Thr Leu Met
130 135 140
Ala Leu Phe Pro Leu His Gln Arg Phe Gly Leu Lys Thr Val Ile Ala
145 150 155 160
Ser Ser Tyr Gln Ala Val Ser Gly Ser Gly Gln His Gly Ile Ala Glu
165 170 175
Leu Glu Thr Gln Val Arg Ala Val Val Asp Gly His Pro Val Val Lys
180 185 190
Asn Val Tyr Pro His Gln Ile Ala Phe Asn Leu Leu Pro Gln Ile Asp
195 200 205
Ser Phe Thr Glu Asn Gly Tyr Thr Lys Glu Glu Leu Lys Met Leu Asn
210 215 220
Glu Gly Arg Lys Ile Leu Ser Leu Pro Glu Leu Lys Val Thr Cys Thr
225 230 235 240
Cys Val Arg Val Pro Val Tyr Arg Ser His Ser Ile Ser Val Thr Ala
245 250 255
Gln Phe Glu Lys Pro Val Asp Leu Glu Thr Ala Arg Ser Ala Tyr Glu
260 265 270
Gly Lys Pro Gly Val Ala Leu Met Asp Asn Pro Ala Glu Gly Val Trp
275 280 285
Pro Thr Pro Leu Asp Ser Thr Asn Gly Asp Thr Cys Tyr Val Gly Arg
290 295 300
Met Arg Met Asp Met Ala Ile Asp Asn Ala Leu Thr Leu Trp Val Val
305 310 315 320
Gly Asp Gln Val Arg Lys Gly Ala Ala Leu Asn Ala Val Gln Ile Ala
325 330 335
Lys Leu Leu Val Asn Arg
340
<210> 7
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Met Gly Gly Met Lys Ala Leu Val Lys Thr Gln Ala Gly Pro Gly Leu
1 5 10 15
Glu Leu Met Asp Val Pro Met Pro Glu Val Gly Pro Asn Asp Val Leu
20 25 30
Ile Lys Ile His Lys Thr Ala Ile Cys Gly Thr Asp Leu His Ile Trp
35 40 45
Asn Trp Asp Lys Trp Ala Gln Gln Thr Ile Pro Val Gly Met His Val
50 55 60
Gly His Glu Phe Cys Gly Val Ile Glu Ser Val Gly Ser Ser Val Thr
65 70 75 80
Glu Tyr Lys Pro Gly Glu Ile Val Ser Gly Glu Gly His Ile Val Cys
85 90 95
Gly His Cys Arg Ser Cys Arg Ser Gly Gln Lys His Leu Cys Pro Asn
100 105 110
Thr Lys Gly Val Gly Val Asn Arg Pro Gly Cys Phe Ala Glu Tyr Leu
115 120 125
Ser Ile Pro Gln Asp Asn Val Val Arg Ile His Lys Ser Ile Pro Met
130 135 140
Glu Ile Ala Ser Ile Phe Asp Pro Leu Gly Asn Ala Val His Thr Ala
145 150 155 160
Leu Ser Trp Asp Leu Val Gly Glu Asp Val Leu Ile Thr Gly Ala Gly
165 170 175
Val Ile Gly Cys Met Ala Ala Ala Val Cys Lys Lys Ala Gly Ala Lys
180 185 190
Thr Val Val Ile Thr Asp Ile Asn Asp Phe Arg Leu Gly Leu Ala Lys
195 200 205
Thr Leu Gly Ala Asp Arg Thr Val Asn Val Thr Arg Glu Lys Leu Glu
210 215 220
Asp Val Met Lys Glu Leu Glu Met Thr Glu Gly Phe Asp Val Cys Leu
225 230 235 240
Glu Met Ser Gly Ala Pro Ser Cys Leu Lys Asp Ile Ile Asp Asn Ser
245 250 255
Arg Asn Gly Ala Asn Ile Ser Leu Leu Gly Ile Gln Pro Asp Gly Ser
260 265 270
Ser Ile Glu Trp Asn Lys Phe Ile Trp Lys Gly Leu Lys Met Lys Gly
275 280 285
Ile Tyr Gly Arg Glu Ile Phe Glu Thr Trp His Lys Met Asp Ser Met
290 295 300
Ile Arg Ser Gly Leu Asn Val Ala Pro Ile Ile Thr His Arg Leu Pro
305 310 315 320
Tyr Thr Glu Phe Arg Glu Gly Phe Glu Ala Met Asn Ser Gly Lys Ser
325 330 335
Gly Lys Val Val Leu Asp Trp Ile Val
340 345
<210> 8
<211> 373
<212> PRT
<213> Exkermanella muciniphila (Akkermansia muciniphila)
<400> 8
Met Gln Gln Thr Ala Arg Ala Ala Val Leu Thr Ala Pro Lys Thr Phe
1 5 10 15
Glu Ile Arg Glu Tyr Pro Ile Pro Ala Ile Gly Asp Asp Glu Met Leu
20 25 30
Ile Lys Val Glu Ala Cys Gly Val Cys Gly Thr Asp Gly His Glu Tyr
35 40 45
Asn Arg Asp Pro Phe Gly Leu Cys Pro Val Val Leu Gly His Glu Gly
50 55 60
Thr Gly Glu Ile Val Ala Met Gly Arg Asn Ile Thr Lys Asp Thr Ala
65 70 75 80
Gly Asn Pro Val Ala Leu Gly Asp Lys Ile Val Thr Cys Ile Ile Pro
85 90 95
Cys Gly Thr Cys Asp Ala Cys Leu Asn Thr Pro Ala Arg Thr Asn Leu
100 105 110
Cys Glu Asn Val Gly Val Tyr Gly Leu Met Pro Asp Asp Asp Val His
115 120 125
Leu Asn Gly Tyr Phe Gly Glu Tyr Leu Val Ile Arg Lys Gly Ser Thr
130 135 140
Phe Phe Asn Val Ser Gly Met Thr Leu Asp Gln Arg Ile Leu Val Glu
145 150 155 160
Pro Ala Ala Val Val Val His Ser Leu Glu Arg Ala Lys Ser Thr Gly
165 170 175
Leu Leu Lys Phe Asn Ser Val Val Leu Val Gln Gly Cys Gly Pro Ile
180 185 190
Gly Leu Leu Gln Ile Ala Thr Leu Arg Thr Leu Gly Ile Glu Thr Ile
195 200 205
Ile Ala Val Asp Gly Asn Asp Ser Arg Leu Glu Leu Ala Arg Glu Met
210 215 220
Gly Ala Ser Arg Thr Tyr Asn Phe Thr Arg Tyr Ala Asp Leu Asn Glu
225 230 235 240
Leu Leu Asp Ala Val Lys Lys Asp Asn Gly Gly Arg Leu Ala Asp Phe
245 250 255
Val Phe Gln Cys Thr Gly Val Gly Lys Ala Gly Ala Asn Ala Trp Lys
260 265 270
Phe Val Lys Arg Gly Gly Gly Leu Cys Glu Val Gly Phe Phe Met Asp
275 280 285
Gly Gly Glu Ser Val Ile Asn His His Tyr Asp Leu Cys Asn Lys Glu
290 295 300
Val Thr Ala Val Gly Ser Trp Val Tyr Ser Pro Gln Asp Tyr Pro Thr
305 310 315 320
Thr Phe Asp Phe Leu Lys Arg Ala Tyr Gly Ile Gly Leu Pro Leu Thr
325 330 335
Lys Leu Ile Ser His Arg Phe Lys Leu Asp Glu Ile Ala Glu Ala Leu
340 345 350
Glu Thr Asn Val Gln Met Lys Gly Ile Lys Ile Ala Val Ile Cys Asn
355 360 365
Cys Ser Lys Asn Ile
370
<210> 9
<211> 333
<212> PRT
<213> Exkermanella muciniphila (Akkermansia muciniphila)
<400> 9
Met Ser Glu Asn His Tyr Ala Glu Phe Ser Glu Cys Ser Met Lys Pro
1 5 10 15
Gln Glu Val Leu Glu Tyr Val Ser Gly Pro Ile Pro Val Pro Glu Glu
20 25 30
Gly Glu Val Leu Val Arg Met Lys Ala Ala Pro Ile Asn Pro Ala Asp
35 40 45
Ile Asn Phe Val Gln Gly Val Tyr Gly Leu Lys Pro Val Leu Pro His
50 55 60
Ser Arg Ala Gly Leu Glu Gly Cys Gly Val Val Gln Glu Ser Arg Ala
65 70 75 80
Ala Gly Phe Arg Glu Gly Asp Glu Val Ile Leu Leu Arg Gly Val Gly
85 90 95
Ser Trp Ser Glu Tyr Val Ala Val Pro Ser Val Asn Val Met Lys Leu
100 105 110
Pro Val Lys Val Asp Pro Val Gln Ala Ala Met Leu Lys Val Asn Pro
115 120 125
Leu Thr Ala Leu Arg Met Leu Glu Gly Phe Val Ser Leu Glu Pro Gly
130 135 140
Asp Trp Leu Val Gln Asn Ala Ala Asn Ser Gly Val Gly Arg Cys Ile
145 150 155 160
Ile Gln Leu Ala Arg Glu Met Gly Val Lys Thr Val Asn Phe Val Arg
165 170 175
Arg Pro Asp Glu Leu Arg Asp Glu Leu Thr Ala Leu Gly Ala Asp Leu
180 185 190
Val Val Gly Glu Asp Asp Gly Asp Val Val Lys Asn Thr Leu Ala Arg
195 200 205
Leu Asp Gly Lys Arg Pro Val Leu Ala Ser Asn Ala Val Gly Gly Glu
210 215 220
Ser Ala Leu Arg Leu Met Asp Met Leu Ala Pro Gly Gly Ser Met Val
225 230 235 240
Thr Tyr Gly Ala Met Ser Arg Lys Ser Ile Lys Val Pro Asn Gly Phe
245 250 255
Leu Ile Phe Lys Gly Ile Lys Leu Glu Gly Leu Trp Val Thr Gln Trp
260 265 270
Leu Lys Asn Ala Pro Val Ser Glu Ile Glu Ala Ala Tyr Glu Lys Leu
275 280 285
Ala Arg Leu Met Ala Asp Gly Arg Leu Lys Gln Ala Val Asp Thr Val
290 295 300
Tyr Pro Leu Ser Asp Val Arg Lys Ala Val Glu Lys Ala Gln Glu Glu
305 310 315 320
Phe Arg Ser Gly Lys Lys Val Leu Ser Met Asp Cys Ala
325 330
<210> 10
<211> 1193
<212> PRT
<213> Exkermanella muciniphila (Akkermansia muciniphila)
<400> 10
Met Thr Asp Ser Ser Ile Pro Asp Met Met Ala Glu Ala Arg Arg Gly
1 5 10 15
Gln Trp Thr Asp Gln Gln Leu Ala Ala Lys Ala Val Glu Leu Ala Glu
20 25 30
Ser Ile Leu Lys Gln Ser Asn Ala Gly Met Arg Gly Lys Glu Lys Arg
35 40 45
Gln Ala Gln Gln Met Glu Arg Met Met Asn Asp Pro Ala Gly Lys Ala
50 55 60
Phe Thr Leu Ala Leu Ala Asp Arg Val Phe Arg Pro Ser Ser Pro Val
65 70 75 80
Arg Gly Ala Glu Leu Phe Arg Tyr Leu Leu Asp Gly Tyr Gly Val Pro
85 90 95
Arg Tyr Leu Ser Ala Ala Asp Arg Phe Ala Ile Lys Met Gly Gly Arg
100 105 110
Phe Ser Ala Gln Phe Pro Gly Val Val Met Pro Val Ile Thr Ser Gln
115 120 125
Leu Arg Lys Glu Ser Ser Asn Val Ile Leu Pro Ala Glu Asp Gly Lys
130 135 140
Leu Arg Pro His Leu Arg Arg Arg Arg Lys Gly Gly Ile Arg Met Asn
145 150 155 160
Ile Asn Gln Leu Gly Glu Ala Ile Leu Gly Glu Ser Glu Ala His His
165 170 175
Arg Leu Gln Gln Val Val Asp Arg Leu Thr Asp Lys Asp Cys Asp Tyr
180 185 190
Ile Ser Val Lys Ile Ser Ala Ile Phe Ser Gln Ile His Leu Val Ala
195 200 205
Phe Glu Glu Thr Val Lys Leu Ile Gln Glu Arg Leu Arg Ile Leu Tyr
210 215 220
Arg Ala Ala Ile Thr Asn Ala Val Thr Leu Pro Asp Gly Ser Arg Lys
225 230 235 240
Pro Lys Phe Val Asn Leu Asp Met Glu Glu Tyr Arg Asp Leu His Leu
245 250 255
Thr Ala Glu Ala Phe Lys Arg Thr Leu Met Glu Asp Glu Phe Met Gln
260 265 270
Leu Glu Ala Gly Ile Val Leu Gln Ala Tyr Leu Pro Asp Ser Trp Glu
275 280 285
Glu Gln Met Lys Leu Cys Ala Trp Ala Lys Glu Arg Val Glu Gln Gly
290 295 300
Gly Ala Arg Ile Lys Ile Arg Leu Val Lys Gly Ala Asn Leu Ala Met
305 310 315 320
Glu Lys Val Glu Ala Ser Met His Gly Trp Ala Gln Ala Pro Tyr Gly
325 330 335
Thr Lys Ala Gln Val Asp Ala Asn Tyr Lys Arg Met Leu His Tyr Gly
340 345 350
Cys Met Pro Asp Asn Ala Arg Tyr Val Gln Phe Gly Val Ala Ser His
355 360 365
Asn Leu Phe Asp Leu Cys Tyr Ala Met Leu Leu Arg Glu Arg Glu Gly
370 375 380
Val Arg Asp Gln Val Glu Phe Glu Met Leu Glu Gly Met Ala Asn His
385 390 395 400
Gln Ala Arg Val Ile Arg Gln Ala Ala Asp Gly Leu Leu Leu Tyr Ala
405 410 415
Pro Val Val Leu Lys Glu Asp Phe His Ser Ala Ile Ala Tyr Leu Val
420 425 430
Arg Arg Leu Asp Glu Asn Thr Ser Glu Glu Asn Phe Leu His Asp Leu
435 440 445
Phe Gly Met Thr Pro Gly Ser Arg Ser Trp Glu Val Gln Lys Lys Arg
450 455 460
Phe Leu Lys Ala Cys Gln Glu Lys Asp Glu Val Lys Tyr Gly Pro Asn
465 470 475 480
Arg Thr Gln Asn Arg Ala Ala Asp Pro Ile Gln Pro Ser His Tyr Arg
485 490 495
Asp Ala Phe Ala Asn Glu Arg Asp Thr Asp Trp Ser Leu Arg Gln Asn
500 505 510
Ala Glu Trp Ile Asn Gly Leu Ile Ala Ala Glu Lys Glu Lys Ser Gly
515 520 525
Glu Glu Ile Pro Leu Val Ile Asp Gly Glu Glu Ile Thr Thr Asn Leu
530 535 540
Trp Gly Val Gly Arg Asp Pro Ser Arg His Asn Glu Val Ser Tyr Lys
545 550 555 560
Phe Ala Tyr Ala Asp Phe Asp Gln Val Glu His Ala Leu Val Thr Ala
565 570 575
Asp Arg Val Arg Ser Ser Trp Ala Ser Lys Ser Ile Gly Glu Arg Ala
580 585 590
Glu Ile Leu His Arg Ala Ala Gln Glu Leu Ser Arg Ile Arg Gly Glu
595 600 605
Ala Ile Ala Ala Met Val Arg Asp Ala Gly Lys Ala Pro Thr Glu Ala
610 615 620
Asp Val Glu Val Ser Glu Ala Ile Asp Phe Cys Arg Tyr Tyr Ala Glu
625 630 635 640
Gly Leu Asp Arg Asp Gly Met Asn Asp Gly Val Glu Met Ser Pro Leu
645 650 655
Gly Ile Ile Cys Val Met Ser Pro Trp Asn Phe Pro Phe Ala Ile Pro
660 665 670
Thr Gly Gly Val Ala Ala Ala Leu Met Ala Gly Asn Ala Val Val Phe
675 680 685
Lys Pro Ser Glu Leu Ala Val Tyr Thr Ala Trp Gln Ile Val Gln Ala
690 695 700
Phe Trp Arg Ala Gly Val Pro Lys Asn Val Leu Gln Phe Val Pro Met
705 710 715 720
Pro Arg Asn Glu Ile Ser Cys Lys Phe Leu Met Asp Pro Arg Leu Asn
725 730 735
Gly Val Ile Met Thr Gly Ser Tyr Arg Thr Gly Lys Met Leu Arg Glu
740 745 750
Leu Arg Pro Asp Leu His Val Leu Ala Glu Thr Ser Gly Lys Asp Ala
755 760 765
Met Ile Ile Thr Ala Thr Ala Asp Pro Asp Gln Ala Val Lys Asp Leu
770 775 780
Val Lys Ser Ala Phe Gly His Ser Gly Gln Lys Cys Ser Ala Ala Ser
785 790 795 800
Val Ala Ile Val Glu Ala Ser Val Tyr Asp Asn Pro Ala Phe Leu Arg
805 810 815
Gln Leu Lys Asp Ala Ala Ala Ser Leu Lys Val Gly Gly Ser Trp Glu
820 825 830
Val Asn Ser Val Val Thr Pro Leu Ile Arg Glu Pro Glu Gly Asn Leu
835 840 845
Leu Arg Ala Leu Thr Gln Leu Glu Pro Gly Glu Glu Trp Leu Leu Lys
850 855 860
Pro Glu Pro Ser Glu Asp Asn Pro Cys Leu Trp Ser Pro Gly Ile Arg
865 870 875 880
Leu Gly Val Lys Pro Gly Ser Trp Phe His Gln Thr Glu Cys Phe Gly
885 890 895
Pro Val Leu Gly Ile Ile Arg Ala Glu Asn Leu Glu Glu Ala Ile Asp
900 905 910
Ile Gln Asn Asp Ser Glu Phe Gly Leu Thr Gly Gly Leu Gln Ser Leu
915 920 925
Asp Glu Arg Glu Ile Ala Leu Trp Lys Thr Lys Val Gln Val Gly Asn
930 935 940
Ala Tyr Ile Asn Arg Val Ile Thr Gly Ala Ile Val Arg Arg Gln Pro
945 950 955 960
Phe Gly Gly Trp Asn His Ser Ser Met Gly Pro Gly Ala Lys Ala Gly
965 970 975
Gly Glu Asn Tyr Leu Thr Met Leu Gly Ser Trp Glu Glu Lys Ala Leu
980 985 990
Pro Gln Lys Leu Arg Thr Pro Gly Glu Arg Ile Ser Gly Leu Val Glu
995 1000 1005
Lys Leu Cys Ser Glu Leu Pro Asp Cys Ala Lys Arg Ile Arg Ser Ala
1010 1015 1020
Ala Gly Ser Gln Ala Lys Trp Trp Met Glu Glu Phe Gly Val Asp His
1025 1030 1035 1040
Asp Pro Ser Arg Val Tyr Gly Glu Asn Asn Thr Phe Arg Tyr Ile Pro
1045 1050 1055
Val Lys Gly Ile Leu Ala Arg Val Glu Asn Met Ser Asp Asp Asp Val
1060 1065 1070
Ala Ile Leu Leu Leu Gly Ala Lys Leu Cys Gly Val Leu Leu His Leu
1075 1080 1085
Ser Ile Gly Thr Ser Arg Pro Trp Ile Gln Lys Met His Gly Tyr Tyr
1090 1095 1100
Ala Ser Leu Thr Val Glu Thr Glu Ala Glu Leu Ile Gly Arg Met Pro
1105 1110 1115 1120
Glu Ala Leu Pro Gly Ile Arg Phe Leu Arg Gly Thr Asp Ile Ser Glu
1125 1130 1135
Thr Leu Ala Asn Ala Ala Arg Ala Arg Asp Val Glu Val Leu Asp Arg
1140 1145 1150
Pro Val Leu Ala Asn Gly Arg Leu Glu Leu Leu Gly Tyr Phe Arg Glu
1155 1160 1165
Gln Ser Val Ser Glu Thr Val His Arg Tyr Gly Asn Leu Ile Pro Pro
1170 1175 1180
Pro Gly Ser Phe Lys Thr Asp Ser Val
1185 1190
<210> 11
<211> 429
<212> PRT
<213> Akkermansia muciniphila (Akkermansia muciniphila)
<400> 11
Met Lys Met Glu Asn Pro Leu Glu Pro Ala Gly Arg Ala Ala Leu Ala
1 5 10 15
Ala Ser Arg Glu Met Leu Ala Leu Glu Pro Glu Glu Lys Ala Gln Ala
20 25 30
Leu Arg Leu Met Ala Ala Gly Val Arg Asn Ala Ala Glu Lys Ile Ile
35 40 45
Arg Glu Asn Val Leu Asp Met Glu Glu Ala Arg Lys Ser Gly Arg Asn
50 55 60
Ser Ala Phe Leu Asp Arg Leu Leu Leu Thr Pro Asp Arg Val Glu His
65 70 75 80
Met Ala Cys Gly Ile Glu Gln Val Ala Ala Leu Pro Asp Pro Val Gly
85 90 95
Glu Cys Ile Arg Glu Trp Thr Arg Pro Asn Gly Leu Arg Ile Arg Gln
100 105 110
Val Arg Val Pro Leu Gly Val Ile Gly Phe Ile Tyr Glu Ser Arg Gly
115 120 125
Thr Val Thr Cys Asp Ala Ala Ala Leu Cys Leu Lys Ser Gly Asn Ala
130 135 140
Val Ile Leu Arg Gly Gly Ser Glu Ser Leu Arg Thr Asn Arg Val Leu
145 150 155 160
Ala Glu Thr Leu Arg Ser Ala Leu Glu Lys Ser Ala Val Pro Ala Asp
165 170 175
Ala Val Arg Leu Leu Asp Ser Gly Ser Arg Asp Glu Val Arg Gln Leu
180 185 190
Cys Glu Leu Asp Ser Cys Leu Asn Val Ile Ile Pro Arg Gly Gly Lys
195 200 205
Gly Leu Ile Arg Ala Val Thr Glu Phe Ser Lys Val Pro Val Leu Lys
210 215 220
His Leu Asp Gly Ile Cys His Val Tyr Val Asp Ala Ala Ala Asp Leu
225 230 235 240
Glu Met Ala Val Asn Val Leu Asp Asp Ala Lys Thr Gln Arg Pro Gly
245 250 255
Val Cys Asn Ala Ala Glu Thr Leu Leu Val Asp Ala Ala Ala Ala Glu
260 265 270
Arg Phe Leu Pro Met Ala Ala Glu Arg Met Arg Met Arg Gly Val Glu
275 280 285
Cys Arg Val Cys Glu Arg Ser Arg Pro Phe Phe Gly Gln Glu Ala Val
290 295 300
Pro Ala Glu Glu Arg Asp Trp Ser Thr Glu Tyr Glu Asp Leu Ile Leu
305 310 315 320
Ser Val Lys Val Val Asp Gly Val Arg Asp Ala Val Glu His Ile Asn
325 330 335
Arg Tyr Gly Ser His His Ser Asp Ser Ile Ile Thr Glu Asp Lys Arg
340 345 350
Ala Arg Asp Tyr Phe Met Asn Arg Val Asp Ser Ala Cys Val Tyr His
355 360 365
Asn Cys Ser Thr Arg Phe Ser Asp Gly Glu Glu Phe Gly Phe Gly Ala
370 375 380
Glu Ile Gly Ile Gly Thr Asp Lys Phe His Ala Arg Gly Glu Met Ala
385 390 395 400
Leu Arg Glu Leu Thr Ser Tyr Lys Tyr Val Ile Glu Gly Asn Gly Gln
405 410 415
Leu Lys Glu Pro Ser Arg Ile Pro Gly Glu Ser Arg Ala
420 425
<210> 12
<211> 336
<212> PRT
<213> Exkermanella muciniphila (Akkermansia muciniphila)
<400> 12
Met Asn Gln Ala Pro His Val Ala Ile Val Gly Ala Thr Gly Ala Val
1 5 10 15
Gly Val Glu Ile Leu Ser Cys Leu Glu Thr Arg Asn Phe Pro Val Gly
20 25 30
Ser Leu Lys Leu Leu Ala Ser Ala Arg Ser Ala Gly Lys Gln Val Ala
35 40 45
Phe Arg Gly Lys Met Leu Thr Val Glu Glu Leu Thr Glu Lys Ser Phe
50 55 60
Asp Gly Val Asp Ile Ala Leu Phe Ser Ala Gly Gly Gly Ile Ser Leu
65 70 75 80
Lys Phe Ala Pro Ile Ala Ala Ala Ala Gly Cys Val Val Ile Asp Asn
85 90 95
Ser Ser Ala Phe Arg Gln Glu Pro Asp Val Pro Leu Val Val Pro Glu
100 105 110
Ile Asn Pro Glu Ala Ala Phe Asn His Pro Arg Asn Ile Ile Ala Asn
115 120 125
Pro Asn Cys Thr Thr Ile Ile Thr Leu Met Ala Leu Phe Pro Leu His
130 135 140
Gln Arg Phe Gly Leu Lys Thr Val Ile Ala Ser Ser Tyr Gln Ala Val
145 150 155 160
Ser Gly Ser Gly Gln His Gly Ile Ala Glu Leu Glu Thr Gln Val Arg
165 170 175
Ala Val Val Asp Gly His Pro Val Val Lys Asn Val Tyr Pro His Gln
180 185 190
Ile Ala Phe Asn Leu Leu Pro Gln Ile Asp Ser Phe Thr Glu Asn Gly
195 200 205
Tyr Thr Lys Glu Glu Leu Lys Met Leu Asn Glu Gly Arg Lys Ile Leu
210 215 220
Ser Leu Pro Glu Leu Lys Val Thr Cys Thr Cys Val Arg Val Pro Val
225 230 235 240
Tyr Arg Ser His Ser Ile Ser Val Thr Ala Gln Phe Glu Lys Pro Val
245 250 255
Asp Leu Glu Thr Ala Arg Ser Ala Tyr Glu Gly Lys Pro Gly Val Ala
260 265 270
Leu Met Asp Asn Pro Ala Glu Gly Val Trp Pro Thr Pro Leu Asp Ser
275 280 285
Thr Asn Gly Asp Thr Cys Tyr Val Gly Arg Met Arg Met Asp Met Ala
290 295 300
Ile Asp Asn Ala Leu Thr Leu Trp Val Val Gly Asp Gln Val Arg Lys
305 310 315 320
Gly Ala Ala Leu Asn Ala Val Gln Ile Ala Thr Leu Leu Val Asn Arg
325 330 335

Claims (9)

1. A protein composition, wherein said protein composition comprises a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and has the activity of the second type of recombinant protein.
2. Use of a protein composition for the preparation of a product for metabolizing ethanol;
the protein composition comprises a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and has the activity of the second type of recombinant protein.
3. The use of claim 2, wherein the first type of recombinant protein is used to metabolize ethanol to acetaldehyde;
the second type of recombinant protein is used to metabolize the acetaldehyde obtained into acetic acid.
4. Use of a host expressing a protein composition for the preparation of a product for metabolizing ethanol.
5. A nucleic acid vector comprising a backbone vector and a polynucleotide encoding a protein composition.
6. A recombinant host for expressing the protein composition, wherein the recombinant host is a microorganism or a cell line; the recombinant host expresses a protein composition.
7. A product comprising at least one of the following components:
a. a protein composition comprising a first type of recombinant protein and a second type of recombinant protein;
the first type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3, or one or more of the amino acid sequences set forth in SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO: 3 by substituting, deleting or adding one or more amino acids and has the activity of the first type of recombinant protein;
the second type of recombinant protein comprises the amino acid sequence shown as SEQ ID NO: 4. SEQ ID NO: 5 and SEQ ID NO: 6, or one or more of the amino acid sequences shown in SEQ ID NOs: 4. SEQ ID NO: 5 and SEQ ID NO: 6 by substitution, deletion or addition of one or more amino acids, respectively, and has the activity of the second type of recombinant protein.
b. A host expressing the protein composition of a;
c. b a fermentation product of said host;
d. b an extract of said host and/or c said fermentation product;
e. other substances having biological activity of metabolizing alcohol.
8. The use of the product of claim 7 in the preparation of anti-hangover drugs/health products.
9. A pharmaceutical composition comprising a protein composition as an active ingredient, and optionally a pharmaceutically acceptable carrier, excipient, adjuvant and/or diluent.
CN202210197701.3A 2022-03-02 2022-03-02 Protein composition and use thereof Active CN114621936B (en)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
CN1706479A (en) * 2004-06-08 2005-12-14 美国酶医公司 Methods and compositions for accelerating alcohol metabolism
US20210052674A1 (en) * 2017-09-28 2021-02-25 Kobiolabs, Inc. Composition for diagnosis and treatment of alcoholic liver disease, using change in short-chain fatty acid producing gut bacterial community

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1706479A (en) * 2004-06-08 2005-12-14 美国酶医公司 Methods and compositions for accelerating alcohol metabolism
US20210052674A1 (en) * 2017-09-28 2021-02-25 Kobiolabs, Inc. Composition for diagnosis and treatment of alcoholic liver disease, using change in short-chain fatty acid producing gut bacterial community

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Title
CHRISTOPH GRANDER 等: "Recovery of ethanol-induced Akkermansia muciniphila depletion ameliorates alcoholic liver disease", GUT, vol. 67, no. 5, pages 891 - 900 *
CHRISTOPH GRANDER 等: "The role of gut vascular barrier in experimental alcoholic liver disease and A. muciniphila supplementation", GUT MICROBES, vol. 12, pages 1851986 *
D. CHENG 等: "A review of a potential and promising probiotic candidate—Akkermansia muciniphila Get access Arrow", JOURNAL OF APPLIED MICROBIOLOGY, vol. 130, no. 6, pages 1813 - 1822 *
无: "Alcohol dehydrogenase zinc-binding domain protein [Akkermansia muciniphila ATCC BAA-835]", GENBANK: ACD05015.1 *
无: "Alcohol dehydrogenase zinc-binding domain protein [Akkermansia muciniphila ATCC BAA-835]", GENBANK: ACD05925.1 *
无: "Aldehyde Dehydrogenase [Akkermansia muciniphila ATCC BAA-835]", GENBANK: ACD05876.1 *
无: "aspartate-semialdehyde dehydrogenase [Akkermansia muciniphila ATCC BAA-835]", GENBANK: ACD05090.1 *
无: "gamma-glutamyl phosphate reductase [Akkermansia muciniphila ATCC BAA-835]", GENBANK: ACD04366.1 *
无: "L-threonine 3-dehydrogenase [Akkermansia muciniphila ATCC BAA-835]", GENBANK: ACD04076.1 *
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